Acidification Enhances Hybrid N₂O Production Associated with Aquatic Ammonia-Oxidizing Microorganisms

Ammonia-oxidizing microorganisms are an important source of the greenhouse gas nitrous oxide (N₂O) in aquatic environments. Identifying the impact of pH on N₂O production by ammonia oxidizers is key to understanding how aquatic greenhouse gas fluxes will respond to naturally occurring pH changes, as well as acidification driven by anthropogenic CO₂. We assessed N₂O production rates and formation mechanisms by communities of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in a lake and a marine environment, using incubation-based nitrogen (N) stable isotope tracer methods with ¹⁵N-labeled ammonium (¹⁵[Formula: see text]) and nitrite (¹⁵[Formula: see text]), and also measurements of the natural abundance N and O isotopic composition of dissolved N₂O. N₂O production during incubations of water from the shallow hypolimnion of Lake Lugano (Switzerland) was significantly higher when the pH was reduced from 7.54 (untreated pH) to 7.20 (reduced pH), while ammonia oxidation rates were similar between treatments. In all incubations, added [Formula: see text] was the source of most of the N incorporated into N₂O, suggesting that the main N₂O production pathway involved hydroxylamine (NH₂OH) and/or [Formula: see text] produced by ammonia oxidation during the incubation period. A small but significant amount of N derived from exogenous/added ¹⁵[Formula: see text] was also incorporated into N₂O, but only during the reduced-pH incubations. Mass spectra of this N₂O revealed that [Formula: see text] and ¹⁵[Formula: see text] each contributed N equally to N₂O by a "hybrid-N₂O" mechanism consistent with a reaction between NH₂OH and [Formula: see text], or compounds derived from these two molecules. Nitrifier denitrification was not an important source of N₂O. Isotopomeric N₂O analyses in Lake Lugano were consistent with incubation results, as ¹⁵N enrichment of the internal N vs. external N atoms produced site preferences (25.0-34.4‰) consistent with NH₂OH-dependent hybrid-N₂O production. Hybrid-N₂O formation was also observed during incubations of seawater from coastal Namibia with ¹⁵[Formula: see text] and [Formula: see text]. However, the site preference of dissolved N₂O here was low (4.9‰), indicating that another mechanism, not captured during the incubations, was important. Multiplex sequencing of 16S rRNA revealed distinct ammonia oxidizer communities: AOB dominated numerically in Lake Lugano, and AOA dominated in the seawater. Potential for hybrid N₂O formation exists among both communities, and at least in AOB-dominated environments, acidification may accelerate this mechanism.